Anti-inflammatory compositions comprising 16alpha-chloromethyl-delta1, 4-pregnadiene-11beta, 21-diol-3, 20-dione



14 Claims. ci. 167-65) The present invention relates to new anti-inflammatory agents, methods of producing the same and methods of using the same, and more particularly to 16- chloromethyl A pregnadiene 11,8,21 diol 3,20- dione and 21-esters thereof which are highly effective anti-inflammatory agents and to methods of producing and using the same.

It is a primary object of the present invention to provide for the production of compounds having an unexpectedly high anti-inflammatory action.

It is another object of the present invention to provide for methods of producing the anti-inflammatory compounds of this invention.

It is yet another object of the present invention to provide compositions for and a method of treating conditions requiring an anti-inflammatory agent in order to achieve a highly effective anti-inflammatory response.

With the above and other objects in view, the antiinflammatory compounds of the present invention mainly comprise 16oz chloromethly A pregnadiene- 11fl,21-diol-3,20-dione and its 21-esters, most particularly the lower aliphatic carboxylic acid 21-esters.

The compositions of the present invention comprise the above compounds as active ingredient plus a pharmaceutical carrier for internal administration, for example by injection or perorally, or :for local administration, for example in the form of a salve or lotion.

The method of achieving an anti-inflammatory effect mainly comprises the administration to a patient requiring an anti-infiammatory action a compound selected from the group consisting of 16a-chloromethyl-A -pregnadiene-l1B,21-diol-3,20-dione and 21-esters thereof.

The method of the present invention of producing the 16a chlonomethyl A pregnadiene l1fi,2ldiol-3,20 dione and its 21-esters mainly comprises the introduction into the molecule of 16a-chloromethyl-A pregnene-l1fl,2l-diol-3,20-dione or its 21-ester, either by pure chemical means or by biochemical means, a l-position double bond. The biochemical method is preferably accomplished by the action of dehydrases of strains of the Bacillus lentus or Corynebacterium simplex.

If the 1-position double bond is introduced into 16a chloromethyl A pregnene 1113,21 diol 3,20- dione, then the 21-ester of the formed l6ot-chloromethyl- N -pregnadiene-l1B,21-diol-3,20-dione is formed -by reaction of the 2l-alcohol with the desired acid so as to form the 21-ester in the manner known in steroid chemistry.

16a methyl A pregnadiene 115,21 diol 3,20- dione, and partciularly its 21-esters, have been found to exhibit a high anti-inflammatory activity. However, quite surprisingly, it has been found that the corresponding 16oz chloromethyl A pregnadiene 115,21-

3,271,252 Patented Sept. 6, 1966 diol-3,20-dione, and its 21-esters, most particularly the 2l-acetate, posess an anti-inflammatory action to a much greater extent than the 16tx-methyl compound. This is proved by comparison of actions by means of the local granuloma-pouch test, the results of which are summarized in the table below.

The test was carried out on male rats (Sprague-Daw- 1y) Weighing 130160 g. Ten animals were used for each dose tested.

For each test, a stock solution of mg. of the substance to be tested in 200 cc. of a benzyl benzoate-castor oil mixture (2:8) was prepared (0.1 mg. in 0.2 cc.). From the stock solution dilutions of 1:3 (0.03 mg. in 0.2 cc.), 1:10 (0.01 mg. in 0.2 cc.), 1:30 (0003 mg. in 0.2. cc.), 1:100 (0.001 mg. in 0.2 cc.), and 1:300 (0.0003 mg. in 0.2 cc.) were used.

The test was carried out by introducing a 25 cc. air bubble by means of a small tube under the skin of the hack. 0.5 cc. of onot-on oil solution (1% in sesame oil were introduced through the same tube. Simultaneously, the injection of the test substance was started to the swelling extending over a period of 4 days, injecting 0.2 cc. of the test solution locally per day. On the third day the air was evacuated from the swelling. On the fifth day of the test the animal was killed by chloroform narcosis, and the amount of exudate in the granulomapouch was determined after aspiration in an injection syrmge.

The average amount of exudate in each .dose group per 100 g. of animal weight was calculated. The percent of reduction of the amount of exudate as compared to the untreated controls was graphically set forth using a semilogarithmic system. From this is found that close which gives an attenuation of the exudate formation of 50% as compared to the untreated controls. This is the designation ED found in the table below.

Action with EDaolmgJ reference to Substance animal/day action of hydrocortisoue acetate=1 lfia-methyl-A -preg1iadiene-11fi,2l-

diol-3,20-dione-21-acetate .1 0. 01 6 lfia-chloromethyl-A -pregnadiene- 11B,21-diol-3,20-dione-2l-acetate 0. 002 18 Prednisolone-acetate r 0.005 11 Hydrocortisone-acetate 0. 055 1 The above table shows that the new loot-chloromethyl- A compound, in the form of its acetate, is three times as active as the corresponding 16u-methyl compound.

As indicated above, the l6ot-chloromethyl-A -pregnadiene-l1,8,21-diol-3,20-dione and its 2l-esters are produced by introduction of a l-position double bond in per se known manner, either chemically or biochemically, into l6a-chloromethyl-M-pregnene11fi,21-diol-3,20- dione, or its 21-ester. The formed crude 16u-chloromethyl-A -pregnadiene-llfl,2l-diol-3,2O-dione is purified in normal manner, for example, by recrystallization from organic solvents, and, as also indicated above, if the end product desired is 21-ester, then the same can be produced in per se known manner from the chosen acid.

The l6a-chloromethyl-A -pregnene-11fl,21-dio1-3,20-dione (and its 21-esters) into which the 1-position double bond is introduced in accordance with the present invention, is a new compound not previously described in the literature. This new compound 16a-chloromethyl- A -pregnene-1l,8,21-diol-3,20 dione (X), which is the useful intermediate in producing the final compound of the present invention, can be produced in accordance with the following reaction mechanism from the known The reaction mass thus obtained is diluted with a large amount of methylene chloride and then washed with sodium thiosulfate solution, with dilute hydrochloric acid and with water, then dried and evaporated. The residue is recrystallized from isopropyl ether. The melting point 16,l7a-methylene-A -pregnene-3fl-ol-20-on-3-acetate (I): is 2l72l8 (3.; the yield is 83% of the theoretical.

(3m (3H CH (I10 ('10 (I oI-I, ---;oI-I

A00 A00 A00 1 (I) 5 (II) 5 (III) 01 c1 (RI-I3 CH2QAC (JHZOAC ([30 00 C| 0 Tor-1,01 i T011201 ]--OH;C1

110 IIO IIO- (1v) (V) (VI) Cl 1 1! i/ (lJmoAc (llHioAc GIHZOH ('30 CO |CO O: O: O:

1 (VII) (VIII) (IX) 61 The following example describes the production of 16a-chloromethyl-M-pregnane-11fl,21-diol-3,20-dione (X) and its 21-acetate (XII):

EXAMPLE I (a) 5 0:,6fl-diClll0l'0-l 6,1 7u-methylene-pregnane-3 5-01- 20-0ne-acetate (II) (IJHzOR CO (b) 1 6 a-Chloromethyl-S a,6,B-dichI0r0-pregnane-3 ,B-ol- ZO-One-acetate (III) 96.7 g. of 5a,6fi-dichloro-l6,l7u-methylene-pregnane- 3B-ol-20-one-acetate are dissolved in 1600 cc. of methylene chloride, hydrogen chloride is introduced until saturation, and the reaction mixture is allowed to stand for 24 hours at 20 C. The major portion of the excess hydrogen chloride is drawn off under vacuum, and the methylene chloride solution is subsequently Washed with water until neutral, dried and evaporated. The residue is recrystallized from isopropyl ether. The melting point is 181-182 C.; the yield is of the theoretical.

(c) 16a-chloromethyl-Sa,6,B-dichloro-pregnane-3fl-0l- 20-0ne (IV) 200 g. of 16u-chloromethyl-5u,6fidich1oropregnane- 3fl-ol-20-one-acetate are dissolved in 1750 cc. of dioxane and 1750 cc. of methanol, mixed with 100 cc. of concentrated hydrochloric acid, and heated under refluxing for 3 hours. One-half of the solution is distilled off, the residue is stirred into ice Water, the precipitated reaction product is filtered off under suction, washed until neutral, dried and recrystallized from methanol. The melting point is 158.5-159 cc.; the yield is 90% of the theoretical.

(d) 1 6a-ch lorom'ethyl-S a,6/3-dichl0r0-pregnarte-3 13,21 dil-20-0ne-21-acerate (V) (a) 34.9 g. of 16a-chloromethyl-5a,6B-dichloro-pregnane-3;8-ol-20-one are dissolved in 800 cc. of chloroform, and after the addition of 1 cc. of hydrogen bromide/ glacial acetic acid, 12.8 g. of bromine in 400 cc. of chloroform are added thereto dropwise. The solution is stirred for an additional 15 minutes, diluted with methylene chloride, washed with water until neutral, dried and evaporated under vacuum. The residue is dissolved in 1000 cc. of dry acetone and heated under stirring and refluxing for 15 hours. The solvent is distilled off under vacuum, and the residue taken up in methylene chloride with water. The organic phase is separated, washed with water, dried and evaporated. The residue is recrystallized from ether. The melting point is 129131 C. with decomposition; yield is 65% of the theoretical.

(B) 6 g. of 16a-chloromethyl-5ot,6fl-dichloro-pregnane- 3fi-ol-20-one are dissolved in 66 cc. of tetrahydrofurane mixed with 9 g. of calcium oxide and 9 g. of iodine and 50 cc. of methanol are added thereto dropwise. The reaction mixture is further stirred for one hour, poured into ice water, the resulting precipitate filtered under vacuum, washed with water and dried at 30 C. under vacuum. The crude 21-iodo compound is dissolved in 250 cc. of acetone and heated under refluxing for 8 hours with 24 g. of potassium acetate. The working up proc'eeds as under (d, a). The yield is 40% of the theoretical.

('y) 1 g. of 16a-chloromethyl-5a,6fi-2l-trichloro-pregnane-3B-ol-20-one having a melting point of 167.5- 169" C. (produced from l6ot-chloromethyl-5a,16,8-dichloro-pregnane-3B-ol-ZO-one by chlorination in 2l-position analogously to (d, at)) are dissolved in 50 cc. of acetone and heated with 4 g. of potassium acetate for 18 hours under refluxing. The Working up proceeds as described under (d, a). The yield is 30% of the theoretical.

(c) J 6 a-ch loromethyl-A -pregnene-3 [3,2 I -di0l-2 O-one- 21 -acetate (VI) A chromium-II-chloride solution (produced from 225 g. of chromium-II-chloride, 900 cc. of water, 150 cc. of concentrated hydrochloric acid and 105 g. of zinc dust) are added dropwise under nitrogen to a solution of 61 g. of 16a-chloromethyl-5a,6fi-dichloro-pregnane-3[3,2l-diol- 20-one-21-acetate in 1500 cc. of acetone, and the reaction mixture is stirred for an additional 1 /2 hours. The reaction mixture is then extracted with methylene chloride, washed with water and evaporated. The residue is recrystallized from isopropyl ether. The melting point is 137138 C. the yield is 70% of the theoretical.

(f) 1 6a-chl0r0met/1yl-A -pregnene-2]-0l-3,20-di0ne- ZI-acetate (VIII) (a) 5 g. of 16a-chlorornethyl-A -pregnene-3fi,2l-diol- 20-one-21-acetate (VI) are dissolved in 100 cc. of acetone and oxidized with 5 cc. of 8 N chromosulfuric acid at 05 C. The reaction product (XIII) is precipitated from water, filtered off under suction, washed until neutral, dried and recrystallized from isopropyl ether-methylene chloride. The melting point is 173174 C.; the yield is 80% of the theoretical.

([3) 10.5 g. of 16a-chloromethyl-5a,6;3-dichloropregnane-3fl,21-diol-20-one-2l-acetate (V) are dissolved in 6 253 cc. of acetic acid and there is added thereto dropwise at 5 C. a chromic acid solution (2.2 g. of chromium trioxide, 2 cc. of water and 23 cc. of glacial acetic acid). The reaction mixture is subsequently mixed with 1.2 cc. of concentrated sulfuric acid, stirred for one hour at 5 C., poured into ice water, the resulting precipitate filtered off under suction, washed, and dried and recrystallized from isopropyl ether. The thus obtained 16achloromethyl 50;,65 dichloro pregnane 21 ol- 3,20-dione-2l-acetate (VII, no definite melting point) is dissolved in 200 cc. of acetone and reacted with chromium-II-chloride solution, the solution described in Example e. The 16a-chloromethyl-A -pregnene-21-ol-3,20- dione-acetate (VIII) is recrystallized from isopropyl ether-methylene chloride. The melting point is 173 174 C. The yield amounts to 60% of the theoretical.

(g) 16a-chl0rometlzyl-M-pregnene-ZI-0l-3,20- dione (IX) (h) 16a-chl0r0methyl-N-pregnene-I1,6,21-di0l- 3,20-di0r e (X) (a) A stainless steel fermenter having a 50 liter capacity is charged with 30 liters of a nutrient solution of the following composition:

Percent Glucose 4.4 NaNO 0.3 KH PO 0.1 Malt extract 1.0 KCl 0.05 FeSO 0.002 MgSO 0.05 Corn steep 0.5

adjusted to pH 7.0, and sterilized by heating for 40 minutes at 120 C. After cooling it is inocculated with a spore suspension of Curvularia lunata (NRRL strain 1, 2380) which is obtained -by rinsing a 7 day corn culture (15 g. corn) with about cc. of physiological sodium chloride solution.

After two days of growth (48 hours) at 25 C. under stirring (220 revolutions/minute) and aeration (1.65 rnfi/hour), 1.8 liters Otf a resulting culture are removed under sterile conditions and transferred into a 50 liter capacity fermenter charged with 28.2 liters of a nutrient solution sterilized as above and containing the following:

Percent Glucose 4.4 Malt extract 1.0 NaNO 0.3 KH PO 0.1

adjusted to pH 7.0.

Alternatively, the main fermenter can be charged with a medium of 10% molasses adjusted to pH 7.0.

After 24 hours of cultivation under stirring (220 revolutions/minute) and aeration 1.65 m. /hour), 7.5 g. of 16a-chlorornethyl-d pregnene-Z1-ol-3,20-dione (IX) in 200 cc. of ethanol are added thereto and the fermentation is continued under the same conditions for 32 hours. The course of the fermentation is checked by removing samples which are extracted with methylisobutyl ketone.

The extract is analyzed by paper chromatography in a system of dioxane-toluene-propylene glycol.

After the end oi the fermentation, the culture broth is filtered off under suction over a 50 liter Buchner funnel and in a vessel provided with a stirrer, stirred two times, each time with 10 liters of methylisobutyl ketone. The extract is concentrated in a vacuum evaporator and in a flask carefully evaporated to dryness under vacuum.

The residue is subjected to chromatography on silica gel. 4.3 g. of a crude oily-crystalline product is obtained with chloroform-ethyl acetate (3:1), and this product is recrystallized several times from ethyl acetatezisopropyl ether. There is thus obtained 1.5 g. of l6a-chloromethyl- M-pregnene-l1/8,21-diol-3,20-dione (X), having a melting point of 176/177-179" C.

(18) Under the conditions described in Example (11, a), 7.5 g. of 16a-chloromethyl-A -pregnene-21-ol-3,20- dione-21acetate (VIII), instead of the free compound, are introduced and fermented for 38 hours.

There is thus obtained 2.5 g. of l6a-chloromethyl-A pregnene-llfi,2l-diol-3,20 dione (X), having a melting point of 177-179 C.

(i 16a-chl0r0methyl-M-pregnene-I1fi,21-di0l-3,20- dione-21 -acetate (XII) 619 mg. of l6a-chloromethyl-M-pregnene-115,21-diol- 3,20-dione (X) are acetylated in 2 cc. of pyridine with 1 cc. of acetanhydride for 2 hours at room temperature. The solution is stirred into 40 cc. of 8% sulfuric acid at C. The precipitate is filtered off under suction, dried and recrystallized from ethanol. The melting point is l51/152153 C.; the yield is 464 mg.

EXAMPLE II I dot-chloromethylA -pregnadiene- 11;8,21-die0l,3,20-di0ne A 50 liter stainless steel fermenter is charged with 30 liters of a nutrient solution containing:

Percent Yeast extract 1.0

Corn steep liquor 5.0

Glucose 2.0

and the pH is adjusted to 7.0.

The nutrient solutioin is sterilized by heating for 40 minutes at 120 C., and after cooling is inocculated with a bacteria suspension of Bacillus lentus which is obtained by rinsing a bouillon agar surface of 64 cm? with 7 cc. of physiological salt solution.

After 24 hours of propagation at 25 C. under stirring (220 revolutions/minute) and aeration (1660* liters/ hour), 1.8 liters of the resulting culture are removed under sterile conditions and transferred into a fermenter containing 28.2 liters of the same medium. At the same time it is mixed with a solution of 7.5 g. of 16a-chloromethyl-M-pregnene-l1B,21-diol-3,20-dione (X) and fermented under the same conditions. The fermentation time amounts to 44 hours.

The course of the fermentation is checked by removal of samples which are extracted with methylisobutyl ketone. The extracts are analyzed by paper chromatography or by polarography. After the expiration of the fermentation time, the culture broth is extracted three times, each time with liters of methylisobutyl ketone. The purified extracts are concentrated in a vacuum evaporator, and then evaporated to dryness in vacuum under nitrogen. The residue is subjected to chromatography on silica gel. 1.62 g. of oily 16u-chloromethyl-A -pregnadiene-115,21- diol-3,20-dione are eluated with chloroform-ethyl acetate mixture. The crude product is recrystallized from methylene chloride/isopropyl ether. The melting point is 70 C.

8 EXAMPLE 111 Under the same conditions as described in Example II, a nutrient solution of 1.0% glucose and 0.2% of corn steep in a 50 liter fermenter is inocculated with bacteria suspension of Corynebacterium simplex, which is obtained by rinsing a bouillon agar surface having an area of 64 cm. with 7 cc. of physiological salt solution, and propagation is continued for 24 hours.

Simultaneously with the transfer of 1.8 liters into a fermenter containing 28.2 liters of the same medium, the mass is mixed with 7.5 g. of 16a-chloromethylcorticosterone (X) in 200 cc. of ethanol and is subjected to fermentation under the same conditions. The fermentation time amounts to 8 hours.

There is thus obtained 1.1 g. of 16a-chloromethyl- A -pregnadiene 11fi,2l-diol-3,20 dione. The melting point is 70 C.

The reaction control and the working up proceeds as described in Example II.

EXAMPLE IV 1 6u-chloromethyl-A -pregnadiene-115321-11701- 3,20-di0ne-21 -acetate 450 mg. of 16a-chloromethyl-A -pregnadiene-11fl,20- diol-3,20-dione are acetylated and further worked up as described in Example I (i). The crude product is recrystallyzed from methylenechloride-isopropyl ether. The melting point is C.

Other 2 1-esters, particularly of the lower aliphatic carboxylic acids, such as propionic acid, butyric acid, etc. can be produced in analogous manner.

EXAMPLE V This example and Example VI illustrate the preparation of compositions for internal administration and for local administration using the active anti-inflammatory agents of the present invention.

Composition for a single tablet:

are formed into a tablet in the usual manner using a tablet press. The diameter of the tablet is 7 mm, the hardness 4 kg. (Stokes hardness tester), and decomposition time in water to 20 C. is 1.5 minutes.

The tablets may be made to contain as little as 1.0 mg. and as much as 5.0 mg. of the active ingredient.

EXAMPLE VI This example illustrates the production of a salve containing 2% by weight of the active ingredient.

Production of kg. of salve:

2.000 kg. 16a-chloromethyl-A -pregnadiene415,21-diol- 3,20-dione-2l-acetate, grain size less than 5, are intro-' duced into 12.970 kg. liquid parafiin and subjected to homogenization two times in homogenizator. The resulting mass is introduced under stirring into a 45 C. melt of 20.000 kg. white Vaseline,

5.000 kg. white wax,

5.000 kg. water free wool fat, and

25.000 kg. Amphocerin K.

9 30.000 kg. de-salted water at 45 C. are emulsified with the above fat phase after the addition of 30 g. perfume Chypre 6466 the mass is while stirring cooled and homogenized.

Containers can then be filled with the salve.

Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can by applying current knowledge readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of the invention and, therefore, such adaptations should and are intended to be comprehended within the meaning and range of equivalence of the following claims.

What is claimed as new and desired Letters Patent is:

1. A compound selected from the group consisting of 1606 chloromethyl-A pregnadiene-11[3,2l diol-3,-20'- dione and propionic acid, butyric acid and acetic acid 2 l-esters thereof.

2. A 21-lower aliphatic carboxylic acid ester of 160:- chloromethyl-A -pregnadiene-l 1,6,21-d1'ol-3,20-dione.

3. 16oz chloromethyl-A pregnadiene-l 15,21 diol- 3,20-dione-2l-a-cetate.

4. 16a chloromethyl A -pregnadiene-1 15,21 diol- 3,20-dione.

5. A compound selected from the group consisting of the lower aliphatic carboxylic acid 2l-esters of 16ac-hloromethyl-A pregnene-l 1B,21-dio1-3,20'-dione.

6. 16a chloromethyl-M-pregnene 11 8,21 dio1-3,20- dione-21-acetate.

to be secured by 7. Set-65 -dlCh10I0-16,17a methylene-pregnane-BB-ol- ZO-one-acetate.

8. IGDC-ChIOI'OmGthYI 5a,6fl-dichloro-pregnane 3fl-ol- -one-acetate.

9. 16a chloromethyl 5a,6fl-dichloro-pregnane 3B- ol-20 one.

10. 16a chloromethyl- 6p dichloro-pregnane 3B- 21-diol-20-one-2l-acetate.

11. 16a chloromethyl M-pregnene 318,21-dio1-20- one-21-acetate.

12. chloromethyl M-pregnene 21-ol-3,20-dione- 21acetate.

13. 16c: chloromethyl A -pregnene-21-ol-3,20-dione.

14. Autiinflammatory composition, comprising a pharmaceutical carrier having distributed therethrough an amount sufficient to have an effective anti-inflammatory action of a compound selected from the group consisting of 16a-chloromethyl-A -pregnadiene 11;3,21-diol-3,20- dione and lower aliphatic carboxylic acid 2l-esters thereof.

References Cited by the Examiner UNITED STATES PATENTS 2,705,237 3/1955 Djerassi et al. 260--397.3 2,756,179 7/1956 Fried et al. 51 2,837,464 3/1958 Nobile 19551 2,936,312 5/1960 Babcock et al. 260--397.4 2,937,192 5/1960 C-olton 260397.4 3,161,661 12/1964 Von Werder et al. 260'-397.4

LEWIS GOT IS, Primary Examiner. ELBERT L. ROBERTS, Assistant Examiner. 

14. ANTI-INFLAMMATORY COMPOSITION, COMPRISING A PHARMACEUTICAL CARRIER HAVING DISTRIBUTED THERETHROUGH AN AMOUNT SUFFICIENT TO HAVE AN EFFECTIVE ANTI-INFLAMMATORY ACTION OF A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 16A-CHLOROMETHYL-$1.4-PREGNADIENE-11B,21-DIOL-3,20DIONE AND LOWER ALIPHATIC CARBOXYLIC ACID 21-ESTERS THEREOF. 